Better prognostic biomarkers are urgently needed to identify patients with early-stage cutaneous melanoma who will progress to aggressive disease. In the past grant period, Project II successfully developed the quantitative real-time RT-PCR assay to detect micrometastases not identified by histopathologic assessment of sentinel lymph nodes (SLN). Project II then adapted this assay for analysis of paraffin-embedded SLN specimens, and incorporated this assay as a stratification factor in the international phase III trial (MSLT-II, Project III, and Core C). In the present proposal, Project II will develop and validate clinically relevant prognostic biomarkers by undertaking genomic/epigenomic/mRNA analyses of primary tumor tissue, metastatic tumor tissue, and blood from melanoma patients accrued in the past grant period. The objective is to develop a molecular model that combines tissue-based assays and blood-based serial assays for diagnosis and prognosis. The objectives are further defined in three aims: Aim I, Assessment of prognostic DNA biomarkers in primary melanomas of patients who have undergone SLN biopsy for melanoma. Aim II, Identification of prognostic RNA and DNA biomarkers in nodal and distant metastases of patients who have undergone SLN biopsy for melanoma. Aim III, Identification and assessment of prognostic circulating DNA and circulating tumor-cell biomarkers for detection of melanoma recurrence after SLN biopsy. These three Aims will take advantage of several unique resources: the clinical specimens with clinical follow-up records for patients in the two ongoing international phase III trials (MSLT-I and MSLT-II, Project Ill) and the extensive independent specimen bank/clinical database at the JWCI melanoma center. Results of these molecular biomarker studies will improve treatment selection for patients with early-stage melanoma and increase early detection of recurrent metastatic melanoma during SLN biopsy postoperative follow-up. This approach will help improve melanoma patient management and staging, especially those of early aggressive disease.